Micronutrients in Support to The Carbon Cycle Activate Antioxidant Defences and Reduce Sperm DNA Damage in Infertile Men Attending Assisted Reproductive Technology Programs: Clinical Trial Study

Oxidative stress has been recognized as a main cause of male subfertility with sperm DNA damage affecting fertilization rates, embryo quality and pregnancy rates within assisted reproductive technology (ART) cycles (1). This has triggered efforts to improve the male fertility, and possibly the ART outcomes, by administering oral antioxidants. The latest available Cochrane review on male infertility (2) concluded that although the quality of evidence is weak, oral antioxidants significantly improve the chances of live birth for couples attending fertility clinics, which confirms the primary role of oxidative imbalance in male infertility. The evidence is so far weaker than expected, mostly due to the low quality of available studies. It is very difficult to establish the amount of antioxidants needed in the single subject and too strong supports may result in excess sperm nuclear decondensation and worsening of male reproductive potential (3), which has been defined as "reductive stress"(4). The available products often contain high doses of antioxidants and multiple exposures, including food fortification, may easily lead to excess of antioxidants with possible negative effects (5).

Under physiologic conditions, the oxy-redox balance, besides leveraging on the assumption of reducing substances with diet, is largely based on the endogenous production of reducing power in the form of glutathione (GSH), a reducing tripeptide with an activated sulfhydryl group (SH) able to donate reducing equivalents and reactivate most of the physiologic antioxidants (6). GSH de novo biosynthesis occurs within the one carbon cycle, responsible for DNA methylation and epigenetics, by transulfuration of its end-product, homocysteine, to cysteine. Cysteine is then complexed with glutamate and glycine to form GSH. The ability of the carbon cycle to support DNA methylation is well-known to be regulated by the availability of dietary micronutrients (7) and this may also be true for the induction of GSH synthesis and antioxidant defences. Indeed, the functions of DNA methylation and antioxidant power generation are cross regulated and may respond to the same micronutrients within a homeostatic unit named "methoxistasis"(8). Methyl donors (e.g. folates), vitamins B2, B3 and B12 and zinc are essential to activate the methylations, which in turn result in activation of GSH synthesis. Vitamin B6, zinc and extra cysteines directly feed GSH synthesis and thus, facilitate the methylations. The concept of micronutrients administration in support to DNA methylation and GSH synthesis, is depicted in Figure 1.

The above substances administered to male partners of couples resistant to ART due to a male factor, resulted, differently from what seen with strong oral antioxidants (9), in a significant correction of both sperm DNA fragmentation and nuclear decondensation suggesting that a boost to the natural antioxidant defences may be effective and devoid of rebound effects (10). Many of the treated patients achieved a pregnancy, either spontaneously or following a new ART cycle, and the pregnancies were strongly correlated with the improvement of sperm nuclear condensation. This was expected due to the known ability of these micronutrients to support DNA methylation (7) and the ability of DNA methylation to trigger chromosome compaction (11). The nuclear compaction could also improve the resistance of DNA to oxidative attacks resulting in less DNA fragmentation. Thus, the induction of the endogenous antioxidant system has not been definitively proven. We tested the same micronutrients in a model of varicocele induction in rats (12) and found that the improvements of sperm DNA damage were paralleled by a sharp reduction of lipid peroxidation, which vouches for a strong induction of antioxidant activity. The present study intended to test the same combination of micronutrients in ART-resistant infertile men and confirm that its effect is linked to a true antioxidant induction as previously seen in the animal model.

In total, 51 patients were enrolled and 8 of them quit for personal reasons. Data on semen analysis preand post-treatment were available for all 43 patients completing the study whereas some data concerning TUNEL (n=36), BODIPY, CMA3 and blue aniline (n=25) methods, were missing.

This was a small size explorative study aimed at testing the hypothesis that a nutritional intervention using micronutrients in support to the carbon cycle, is of benefit to infertile men attending ART programs and that it is able to induce both improved DNA methylation and resumption of the endogenous antioxidant activity.

The administration of micronutrients did not modify sperm count or motility but improved sperm morphology, which can be assumed as a possible effect on the processes of DNA methylation that contribute to the epigenetic programming of the cell phenotype. The parallel improvement of sperm nuclear maturation shown by CMA3 and blue aniline staining, also points to a possible effect on DNA methylation. Indeed, DNA and histone methylation plays a pivotal role in sperm nuclear maturation and is also involved in the process of protamination (20). The induction of DNA methylation is explained by the ability of the administered folates to feed homocysteine re-methylation to methionine. Methionine is adenylated to generate the universal methyl donor S-adenosylmethionine (SAMe) acting as a substrate for DNA N-methyltransferases. We also supplemented vitamin B12 that is needed to pass the methyl group from folates to homocysteine, vitamins B2 and B3 as cofactors for methylentetrahydrofolate reductase (MTHFR) to activate folates, and zinc that is as well necessary to MTHFR and methionine synthase for homocysteine remethylation.

Micronutrients supplementation achieved a significant reduction of sperm DNA fragmentation and lipid peroxidation. The improvement of DNA fragmentation was likely of clinical relevance because the average rate moved from 23.2 to 17.8% (i.e. it dropped below the critical threshold of 20% that is assumed as clinically relevant) (21). This may imply an improved performance of the endogenous antioxidant metabolism because the administered micronutrients did not include any direct antioxidant substances. However, the origin of sperm DNA fragmentation is controversial with several processes showing the ability to contribute. According to Muratori et al. (22), oxidative aggression does not behave as the primary trigger, rather it is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and oxidative stress during the transit in the male genital tract. Therefore, the positive effect of micronutrients on DNA fragmentation may be already explained by the protection from an improved chromatin packaging resulting from the induction of methylations. However, an improvement of the antioxidant defences may also be involved.

Micronutrients also achieved a significant drop in sperm lipid peroxidation to half of the baseline value. Lipid peroxidation is a process where oxidants attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs), resulting in production of other radicals attacking other double bonds in a self-amplified process and is a clear-cut oxidative damage (23). The abundance of PUFAs in the sperm membrane is a reason for the particular sensitivity of sperms to lipid peroxidation if an oxidative imbalance occurs (24). GSH is produced as a reaction to oxidative stress by activation of the redox-sensitive transcription factor, nuclear factor erythroid-2-related factor 2 (Nrf2), that activates the enzyme γ-glutamyl cysteine ligase. This enzyme is further regulated by the availability of intracellular cysteines from both the diet and endogenous synthesis (25). Our micronutrients included soluble cysteine in the form of N-acetylcysteine and supported the endogenous cysteine synthesis by zinc and vitamin B6 acting as the necessary co-factor for cystathinonine β-synthase (CBS), the enzyme addressing homocysteine from the carbon cycle to cystathionine and then, cysteine synthesis. These micronutrients had therefore the ability to support antioxidant defences by facilitating GSH synthesis and the reduction of lipid peroxidation was likely a sign of antioxidant defences induction.

In addition, a synergy with other micronutrients is likely to apply. Abundance of SAMe from the carbon cycle can bind CBS resulting in an allosteric activation with a fivefold increase of enzyme activity (26). Thus, the administered micronutrients had the potential to induce a strong boost to intracellular cysteine availability by increasing both its nutritional intake and the endogenous synthesis for a better response of the GSH system to an increased oxidative load and this may account for the effect on lipid peroxidation. Worth to note, the synthesis of cysteine from homocysteine is the only source of the intracellular gasotransmitter H₂S (27). H2S in turn promotes the activation of vitamin B12 (28) and plays as a main inducer of the carbon cycle designing a cross activated homeostatic unit.

Our study was too small in size to provide meaningful clinical findings; however, the outcomes were aligned with a possible positive effect of the treatment on the patients reproductive performance. Four out of 25 (16%) couples opting for an expectant management strategy, achieved a clinical pregnancy during the follow-up period. Interestingly, the male partners of these couples also showed a good response to the micronutrients of the sperm damage indexes (data not shown), which endorses a possible link between the positive pregnancy outcome and the treatment. Out of 18 couples opting for a new ART cycle, only 12 had viable embryos and only 10 of them underwent either fresh (n=4) or thawed (n=6) embryo transfer, which resulted in 6 clinical pregnancies. Again, the male partners of the couples achieving a pregnancy were good responders for sperm parameters. Altogether, these clinical outcomes indicate good chances that the treatment can be of help to male reproductive function at least in good responders. Reasons for lack of response may be bad treatment compliance, a negative genetic background and occurrence of other pathogenic mechanisms beside oxidative aggression, but our data do not allow to further speculate on this.

In summary, the oral administration of a combination of micronutrients including folates, B vitamins, zinc and cysteines to male partners of ART-resistant couples, showed the ability to reduce sperm DNA damage and improve sperm nuclear maturation and the clinical outcomes reflected a positive effect. These outcomes were related to the ability of the micronutrients to activate the one carbon cycle resulting in both stronger methylation ability and activation of the endogenous antioxidant defences. The recorded antioxidant effect, which was strong and likely of clinical value, was achieved without perturbations of the cell homeostasis as happen with oral antioxidants (9).

The present study failed to address a series of relevant questions including the actual clinical gain that can be achieved by micronutrients, how to individuate potential good responders, dose and duration of the treatment and whether the add-on of oral antioxidants may further improve the effect or rather just derange the homeostatic regulations. All of these questions should be addressed by larger size clinical trials centred on the clinical outcomes. Meantime, micronutrients qualify as an alternative to antioxidants in correcting oxidative damage in infertile men.